The Fitness Consequences of Aneuploidy Are Driven by Condition-Dependent Gene Effects

<div><p>Aneuploidy is a hallmark of tumor cells, and yet the precise relationship between aneuploidy and a cell’s proliferative ability, or cellular fitness, has remained elusive. In this study, we have combined a detailed analysis of aneuploid clones isolated from laboratory-evolved populations of <i>Saccharomyces cerevisiae</i> with a systematic, genome-wide screen for the fitness effects of telomeric amplifications to address the relationship between aneuploidy and cellular fitness. We found that aneuploid clones rise to high population frequencies in nutrient-limited evolution experiments and show increased fitness relative to wild type. Direct competition experiments confirmed that three out of four aneuploid events isolated from evolved populations were themselves sufficient to improve fitness. To expand the scope beyond this small number of exemplars, we created a genome-wide collection of >1,800 diploid yeast strains, each containing a different telomeric amplicon (Tamp), ranging in size from 0.4 to 1,000 kb. Using pooled competition experiments in nutrient-limited chemostats followed by high-throughput sequencing of strain-identifying barcodes, we determined the fitness effects of these >1,800 Tamps under three different conditions. Our data revealed that the fitness landscape explored by telomeric amplifications is much broader than that explored by single-gene amplifications. As also observed in the evolved clones, we found the fitness effects of most Tamps to be condition specific, with a minority showing common effects in all three conditions. By integrating our data with previous work that examined the fitness effects of single-gene amplifications genome-wide, we found that a small number of genes within each Tamp are centrally responsible for each Tamp’s fitness effects. Our genome-wide Tamp screen confirmed that telomeric amplifications identified in laboratory-evolved populations generally increased fitness. Our results show that Tamps are mutations that produce large, typically condition-dependent changes in fitness that are important drivers of increased fitness in asexually evolving populations.</p></div

Alternative beneficial mutations are selected in the absence of the main driver.

<p>(<b>A</b>) The copy number of <i>SUL1</i> was assessed using qPCR of samples taken from two independent experiments in which <i>SUL1</i> was not amplified (green and pink) and compared with previously published data from wild-type strains (in grey) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006339#pgen.1006339.ref040" target="_blank">40</a>]. (<b>B</b>) The fitness coefficient as compared to the ancestral strain of population samples at generations 5, 50, and 200 and the fitness of two clones isolated at generation 200. (<b>C</b>) A small deletion (~5kb) encompassing four genes on chromosome IV was detected in a population from one experiment (between brackets); polyT sequences are present at the breakpoints. The colors of the boxes represent the orientation of the genes (yellow: gene on the Watson strand, grey: genes on the Crick strand). (<b>D</b>) Fitness coefficients of the deletion strains <i>ipt1</i>Δ and <i>snf11</i>Δ and those of both deletion strains complemented with <i>IPT1</i> or <i>SNF11</i> on a low-copy plasmid grown in sulfate limitation.</p

Aneuploidy variably affects fitness of evolved clones.

<p>The individual fitness effects of point mutations and aneuploid events were determined for all mutations identified in the evolved clones S8c2 (A), P5c3 (B), and P6c1 (C). The supernumerary chromosome(s) in each clone are labeled by their identifying translocation or, in the case of the chromosome XIII disomy, as “Chr XIII.” Aneuploid and euploid clones are color-coded according to the legend. As described in the text, “All” for P5c3 and P6c1 and “None” for P5c3 represent backcrossed segregants that contain all or none of the mutations present in the original evolved clone. “No <i>SUL1</i> amp” for S8c2 is a backcrossed segregant that contains all the mutations identified in S8c2 except for the <i>SUL1</i> amplicon. Raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s010" target="_blank">S2 Table</a>.</p

Alternative accessible evolutionary paths.

<p><b>(A)</b> The fitness of beneficial mutations found (F) in Evolve and Resequence studies is significantly higher than the fitness of beneficial mutations not found (NF) in sulfate-limitation but not in glucose-limitation. The significance of the difference between the two boxplots for each condition was estimated using a Wilcoxon-ranked test. (<b>B)</b> Each point represents the fitness of a strain and the proportion of Evolve and Resequence samples with the corresponding gene mutated. <i>SUL1</i> dominates the fitness and mutational spectrum. Several mutations have a high fitness but have never been detected in Evolve and Resequence studies and might correspond to potential drivers of adaptation.</p

Experimental design for genome-wide screen for the fitness effects of Tamps.

<p>A) A genome-wide pool of telomeric amplicon strains (Tamps) was constructed. Each Tamp initiates at the <i>KanMX</i> cassette and extends to the proximal telomere, creating a strain that has two chromosomal copies (2N) at most genomic locations, one copy (1N) in the region replaced by the <i>KanMX</i> cassette in the deletion collection, and three copies (3N) at locations telomeric of the deleted gene. The Tamp BC and a portion of KanMX are also present at 2 copies. Each strain contains two barcodes: one identifying the Tamp and a second identifying the unique biological replicate. A third barcode was incorporated during the generation of the barcode sequencing libraries which allowed for multiplexing of experimental samples. Large black arrows represent telomeres; large black circles represent centromeres. The primers represent the barseq primers used to create libraries for sequencing. B) Genome-wide pooled competition experimental design.</p